仿刺参溶菌酶在重组酿酒酵母中的表达  

Expression of Apostichopus japonicas lysozyme in recombinant Saccharomyces cerevisiae

作　　者：随瑞瑞 沈咪娜 李成[1] 赵钰[1] 车明月 张齐[1] 丛丽娜[1] 

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《大连工业大学学报》2017年第3期162-167,共6页Journal of Dalian Polytechnic University

基　　金：辽宁省教育厅一般科学研究项目(L2014217);辽宁省教育厅重点实验室项目(LZ2014029);辽宁省研究生教育创新计划项目(2014-154)

摘　　要：采用酿酒酵母MKP-0表达仿刺参溶菌酶蛋白(AjLys)。以质粒pMD18-AjLys为模板,PCR扩增将组氨酸标签His-tag克隆到AjLys基因的N末端,构建克隆载体pMD18-His6-AjLys。根据密码子偏好性,通过定点突变对AjLys蛋白第69、77和95位的3个精氨酸的密码子进行优化,得到克隆载体pMD18-His6-AjLys(Δ69,77,95)。经SpeⅠ/BglⅡ酶切,构建重组表达质粒pESC-LEU-His6-AjLys和pESC-LEU-His6-AjLys(Δ69,77,95)。基因工程菌pESC-LEU-His6-AjLys(Δ69,77,95)/MKP-0通过半乳糖诱导72h后,Western Blot检测结果表明其成功表达了AjLys蛋白。抑菌实验结果表明,AjLys蛋白对溶壁微球菌具有一定的抑制作用。本实验成功构建了AjLys的酿酒酵母表达载体,并可在MKP-0细胞中表达了具有活性的AjLys蛋白,为AjLys的生产提供了一种具有可行性的新方法。Apostichopus japonicus lysozyme （AjLys） was expressed by Saccharomyces cerevisiae. The His-tag was inserted into the N terminus of AjLys gene based on pMD18-AjLys vector to construct the new plasmid pMD18-His6-AjLys. According to the analysis by Codon Usage Database in S. cerevisiae, AjLys gene was optimized by point mutation to obtain pMD18-His6-AjLys （A69, 77, 95） vector. The recombinant expression vectors pESC-LEU-His6-AjLys and pESC-LEU-His6-AjLys （A69, 77, 95） were constructed. AjLys protein was successfully expressed in the recombinant pESC-LEU-His6-AjLys （A69, 77, 95）/MKP-0 cell inducted by galactose, which was detected by Western Blot. The expressed AjLys protein displayed inhibitive effect on the growth of the Micrococcus lysodeikticus. The result showed that AjLys expression vector of S. cerevisiae was constructed successfully, and the active AjLys protein was expressed in MKP-0 cells, which provided a new method for the production of AjLys.

关 键 词：仿刺参 溶菌酶 酿酒酵母 密码子优化 表达 

分 类 号：TS254.1[轻工技术与工程—水产品加工及贮藏工程] Q556.2[轻工技术与工程—食品科学与工程]